Correction: A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction.

[This corrects the article DOI: 10.1371/journal.pgen.1005810.].

A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction.

At the Drosophila NMJ, BMP signaling is critical for synapse growth and homeostasis. Signaling by the BMP7 homolog, Gbb, in motor neurons triggers a canonical pathway-which modulates transcription of BMP target genes, and a noncanonical pathway-which connects local BMP/BMP receptor complexes with the cytoskeleton. Here we describe a novel noncanonical BMP pathway characterized by the accumulation of the pathway effector, the phosphorylated Smad (pMad), at synaptic sites. Using genetic epistasis, histology, super resolution microscopy, and electrophysiology approaches we demonstrate that this novel pathway is genetically distinguishable from all other known BMP signaling cascades. This novel pathway does not require Gbb, but depends on presynaptic BMP receptors and specific postsynaptic glutamate receptor subtypes, the type-A receptors. Synaptic pMad is coordinated to BMP's role in the transcriptional control of target genes by shared pathway components, but it has no role in the regulation of NMJ growth. Instead, selective disruption of presynaptic pMad accumulation reduces the postsynaptic levels of type-A receptors, revealing a positive feedback loop which appears to function to stabilize active type-A receptors at synaptic sites. Thus, BMP pathway may monitor synapse activity then function to adjust synapse growth and maturation during development.

Immunofluorescence and image analysis pipeline for Drosophila motor neurons.

The neuromuscular junction (NMJ) of larval Drosophila is widely used as a genetic model for basic neuroscience research. The presynaptic side of the NMJ is formed by axon terminals of motor neurons, the soma of which reside in the ventral ganglion of the central nervous system (CNS). Here we describe a streamlined protocol for dissection and immunostaining of the Drosophila CNS and NMJ that allows processing of multiple genotypes within a single staining tube. We also present a computer script called Automated Image Analysis with Background Subtraction which facilitates identification of motor nuclei, quantification of pixel intensity, and background subtraction. Together, these techniques provide a pipeline for neuroscientists to compare levels of different biomolecules in motor nuclei. We conclude that these methods should be adaptable to a variety of different cell and tissue types for the improvement of efficiency, reproducibility, and throughput during data quantification.

The RhoGEF trio functions in sculpting class specific dendrite morphogenesis in Drosophila sensory neurons.

BACKGROUND: As the primary sites of synaptic or sensory input in the nervous system, dendrites play an essential role in processing neuronal and sensory information. Moreover, the specification of class specific dendrite arborization is critically important in establishing neural connectivity and the formation of functional networks. Cytoskeletal modulation provides a key mechanism for establishing, as well as reorganizing, dendritic morphology among distinct neuronal subtypes. While previous studies have established differential roles for the small GTPases Rac and Rho in mediating dendrite morphogenesis, little is known regarding the direct regulators of these genes in mediating distinct dendritic architectures. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that the RhoGEF Trio is required for the specification of class specific dendritic morphology in dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS). Trio is expressed in all da neuron subclasses and loss-of-function analyses indicate that Trio functions cell-autonomously in promoting dendritic branching, field coverage, and refining dendritic outgrowth in various da neuron subtypes. Moreover, overexpression studies demonstrate that Trio acts to promote higher order dendritic branching, including the formation of dendritic filopodia, through Trio GEF1-dependent interactions with Rac1, whereas Trio GEF-2-dependent interactions with Rho1 serve to restrict dendritic extension and higher order branching in da neurons. Finally, we show that de novo dendritic branching, induced by the homeodomain transcription factor Cut, requires Trio activity suggesting these molecules may act in a pathway to mediate dendrite morphogenesis. CONCLUSIONS/SIGNIFICANCE: Collectively, our analyses implicate Trio as an important regulator of class specific da neuron dendrite morphogenesis via interactions with Rac1 and Rho1 and indicate that Trio is required as downstream effector in Cut-mediated regulation of dendrite branching and filopodia formation.

Growing pains: development of the larval nocifensive response in Drosophila.

The ability to perceive and avoid harmful substances or stimuli is key to an organism's survival. The neuronal cognate of the perception of pain is known as nociception, and the reflexive motion to avoid pain is termed the nocifensive response. As the nocifensive response is an ancient and evolutionarily conserved behavioral response to nociceptive stimuli, it is amenable to study in relatively simple and genetically tractable model systems such as Drosophila. Recent studies have taken advantage of the useful properties of Drosophila larvae to begin elucidating the neuronal connectivity and molecular machinery underlying the nocifensive response. However, these studies have primarily utilized the third-instar larval stage, and many mutations that potentially influence nociception survive only until earlier larval stages. Here we characterize the nocifensive responses of Drosophila throughout larval development and find dramatic changes in the nature of the behavior. Notably, we find that prior to the third instar, larvae are unable to perform the characteristic "corkscrew-like roll" behavior. Also, we identify an avoidance behavior consistent with a nocifensive response that is present immediately after larval hatching, representing a paradigm that may be useful in examining mutations with an early lethal phenotype.

Turtle functions downstream of Cut in differentially regulating class specific dendrite morphogenesis in Drosophila.

BACKGROUND: Dendritic morphology largely determines patterns of synaptic connectivity and electrochemical properties of a neuron. Neurons display a myriad diversity of dendritic geometries which serve as a basis for functional classification. Several types of molecules have recently been identified which regulate dendrite morphology by acting at the levels of transcriptional regulation, direct interactions with the cytoskeleton and organelles, and cell surface interactions. Although there has been substantial progress in understanding the molecular mechanisms of dendrite morphogenesis, the specification of class-specific dendritic arbors remains largely unexplained. Furthermore, the presence of numerous regulators suggests that they must work in concert. However, presently, few genetic pathways regulating dendrite development have been defined. METHODOLOGY/PRINCIPAL FINDINGS: The Drosophila gene turtle belongs to an evolutionarily conserved class of immunoglobulin superfamily members found in the nervous systems of diverse organisms. We demonstrate that Turtle is differentially expressed in Drosophila da neurons. Moreover, MARCM analyses reveal Turtle acts cell autonomously to exert class specific effects on dendritic growth and/or branching in da neuron subclasses. Using transgenic overexpression of different Turtle isoforms, we find context-dependent, isoform-specific effects on mediating dendritic branching in class II, III and IV da neurons. Finally, we demonstrate via chromatin immunoprecipitation, qPCR, and immunohistochemistry analyses that Turtle expression is positively regulated by the Cut homeodomain transcription factor and via genetic interaction studies that Turtle is downstream effector of Cut-mediated regulation of da neuron dendrite morphology. CONCLUSIONS/SIGNIFICANCE: Our findings reveal that Turtle proteins differentially regulate the acquisition of class-specific dendrite morphologies. In addition, we have established a transcriptional regulatory interaction between Cut and Turtle, representing a novel pathway for mediating class specific dendrite development.

Isolation and purification of Drosophil peripheral neurons by magnetic bead sorting.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP) under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver or the control of the pan-da neuron-specific GAL4 driver. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.